JP5498005B2 - Process for producing alcohols via organic acids - Google Patents

Description

  The present invention relates to a wide range of saccharides (monosaccharides, oligosaccharides, uronic acids, etc.) obtained by converting lignocellulose directly into an organic acid by a wet oxidation method or hydrolyzing lignocellulose with pressurized hot water. ) By acetic acid fermentation by microorganisms to obtain an organic acid, and the organic acid thus obtained is esterified and hydrocracked to synthesize alcohols such as methanol and ethanol.

  A conventional method for producing ethanol is a method in which a 6-carbon sugar such as glucose is used as a starting point, and this is fermented by yeast Saccharomyces cerevisiae or bacteria Zymomonas mobilis (see Formula I).

Ethanol fermentation from glucose by yeast and bacteria

  In contrast, lignocellulose is converted to organic acids such as formic acid and acetic acid by wet oxidation, or a wide range of saccharides (monosaccharides, oligosaccharides, uronic acids) obtained by hydrolyzing lignocellulose with pressurized hot water. Etc.) by acetic acid fermentation by microorganisms to obtain an organic acid, which is reacted with alcohol to esterify, and then the ester is hydrocracked at high temperature and high pressure in the presence of a catalyst to synthesize alcohols. is there. Examples in the case of synthesizing ethanol after esterifying acetic acid and then hydrocracking it are shown in formulas II, III and IV.

Acetic acid fermentation of glucose when cellulose [C ₆ H ₁₀ O ₅ ] _n is hydrolyzed to give glucose C ₆ H ₁₂ O ₆

  As described above, in the case of ethanol fermentation with yeast, only 2 mol of ethanol can be produced from 1 mol of glucose, whereas when lignocellulose is converted to acetic acid, 3 mol of ethanol can be produced from 1 mol of glucose. In the former, carbon dioxide is released as a by-product, whereas in the latter it is not. From these points, “indirect conversion bioethanol technology”, in which glucose is converted to acetic acid and then ethanol is chemically synthesized, has attracted attention.

  In recent years, research on hydrolyzing lignocellulosic resources, mainly plant-based biomass, to glucose, which is the main constituent unit, and producing various biofinals such as ethanol and lactic acid has been promoted. Although it is present in the largest amount on earth and has a high potential for ethanol production, the reason why its use does not progress slowly is considered to be as follows.

  The first problem is sugar production from lignocellulose. The sugars obtained by acid hydrolysis of lignocellulose are monosaccharides such as 5 and 6 carbon sugars, their oligosaccharides (cellooligosaccharides and xylooligosaccharides), and the acidic sugar residue of xylan, one of hemicelluloses, uron Although there are various types of acids, the conventional yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis can convert only 6 carbon sugars to ethanol. Therefore, an oligosaccharide needs to be converted into a monosaccharide by cellulase or xylanase, and an arming yeast having a cellulase function and a fermentation function has been developed, but it is not general. In addition, ethanol conversion of pentose is also being studied by genetic recombination technology, but its stability and impact on the natural world must be taken into account, and it involves many problems. As a result, the fermentable saccharides in a simple acid saccharification process are limited to only 6 carbon sugars and therefore their yield is limited.

The second problem lies in ethanol fermentation that everyone has taken for granted. As shown in the following formula (V), 2 mol equivalent of ethanol and 2 mol equivalent of carbon dioxide as a by-product are discharged from 1 mol of glucose. Carbon dioxide is carbon neutral, but only 4 of the 6 carbons of 6 carbon sugars have been converted to ethanol. Therefore, this method is not a desirable process from the viewpoint of reducing greenhouse gases.

In the case of 5-carbon sugar, as shown in the following formula (VI), considering that only 3.3 carbons out of 5 carbons can be converted to ethanol, the necessity of genetic recombination technology must be questioned. .

  To summarize these problems, ethanol production from lignocellulose is based on gene recombination technology, sulfuric acid recovery by state-of-the-art column separation using anion exchange resin, and membrane separation using zeolite. Even using ethanol concentration, it is almost hopeless to exceed the highest reported energy efficiency of 40%, and lignocellulose has a technology superior to ethanol production from starch and molasses without introducing innovative technology. Can not expect.

  Based on this idea, the present inventors propose a novel ethanol production process based on esterification of an organic acid containing acetic acid and its hydrocracking in order to enable the current energy efficiency to exceed 40%. . A process for synthesizing ethanol by esterifying acetic acid and then hydrocracking the ester has already been proposed (see Patent Document 1). However, this has the following problems (i) and (ii).

(i) Clostridium bacteria are anaerobic homofermentative bacteria that produce only acetic acid from a substrate such as sugar at an optimum temperature of around 60 ° C. However, the problem is that resistance to acetic acid produced by itself is as low as 1%. As a countermeasure, it is possible to keep acetic acid concentration below 1% by reducing acetic acid inside the fermenter at 60 ° C and continuously distilling acetic acid together with water from the culture solution. Since the boiling point of acetic acid is 118 ° C., which is higher than that of water, it cannot be denied that this method is not efficient. Also, in order to separate and recover acetic acid as a salt by adding alkali salts, this method is not efficient because the concentration of acetic acid in the culture solution is about 1%.

(ii) Patent Document 1 only mentions known hydrogen production methods such as electrolysis of hydrogen derived from petroleum refining processes, water electrolysis, and steam gasification of biomass. In terms of cost, hydrogen, which is a byproduct of the oil refining process, is the cheapest, but it does not match the concept of “bioethanol” in that it uses hydrogen derived from fossil fuels.
Special table 2002-537848 gazette

  It is an object of the present invention to provide a method for producing alcohols that can solve the above problems.

  The present invention is characterized in that lignocellulose is converted into an organic acid by a wet oxidation method or using a microorganism, the organic acid is esterified, and then the ester is hydrocracked to produce an alcohol. This is a method for producing alcohols.

  First, the case where lignocellulose is converted into an organic acid by a wet oxidation method will be described.

  Hydrolysis of lignocellulose, which is the main constituent of herbs and woody plants, is hydrolyzed with pressurized hot water at 140 to 400 ° C to obtain organic acids such as formic acid, lactic acid, and acetic acid. Alcohols such as methanol and ethanol are produced via hydrogenolysis.

  In addition, the biomass is subjected to a wet oxidation reaction in an alkaline high-temperature and high-pressure water of 0.3 M or higher under a relatively mild condition, for example, in the range of 130 to 350 ° C. 75%) and can be converted to formic acid. Thus, formic acid can be obtained from lignocellulose in a high yield. Further, formic acid can be more selectively produced by using hydrogen peroxide for the super-subcritical water treatment of biomass (see JP 2007-39368 A).

  When the organic acid is acetic acid, conventionally, acetic acid and ethanol are mixed at the same molar ratio for esterification, but sulfuric acid or the like is required as a catalyst in addition to acetic acid and ethanol. However, if acetic acid and ethanol are similarly mixed and treated near the supercritical temperature, the esterification proceeds without catalyst and ethyl acetate is synthesized. This eliminates the need to separate the esterification product (ethyl acetate) and sulfuric acid, eliminates the cost of recovery and reuse of sulfuric acid, and avoids corrosion of the reaction vessel (due to sulfuric acid). Arise.

  When the organic acid is formic acid, in the conventional method, formic acid and methanol are mixed in the same molar ratio for esterification, but sulfuric acid or the like is required as a catalyst in addition to formic acid and methanol. However, when formic acid and methanol are mixed in the same manner and treated at around the supercritical temperature, the esterification proceeds without the catalyst and methyl formate is synthesized. This eliminates the need to separate the esterification product (methyl formate) and sulfuric acid, eliminates the cost of recovery and reuse of sulfuric acid, and avoids corrosion (due to sulfuric acid) of the reaction vessel. Arise.

  Next, a case where lignocellulose is converted into an organic acid using a microorganism will be described.

  In this case, lignocellulose is delignified, and the resulting holocellulose is hydrolyzed by low-temperature and high-temperature two-stage pressurized hydrothermal method, and the resulting 5-carbon sugar, 6-carbon sugar, their oligosaccharides, uron Acids and lignin-derived materials are converted to acetic acid by anaerobic homoacetic acetic acid fermentation bacteria of the genus Clostridium that can assimilate them. Since it is not necessary to hydrolyze holocellulose to monosaccharides, hydrolysis with pressurized hot water is effective.

  Substances that can be converted to acetic acid by anaerobic homoacetic acid-fermenting bacteria are usually obtained by hydrolyzing holocellulose obtained by delignification of lignocellulose using a low-temperature and high-temperature two-stage pressurized hydrothermal method. In addition, pentoses, hexoses, their oligosaccharides, uronic acids, and lignin-derived substances, among them, pentoses, hexoses, their oligosaccharides, and uronic acids may be used.

  In the above method, anaerobic hetero acetic acid-fermenting bacteria that can use cellulose as a substrate converts cellulose and oligosaccharides constituting it into hexose, and further converts into acetic acid, formic acid, and lactic acid. It is also preferable to allow cellulose to be converted into acetic acid with higher efficiency by symbiosis with anaerobic homoacetic acid-fermenting bacteria capable of converting lactic acid into acetic acid.

  The substance hydrolyzed by the pressurized hydrothermal method is usually holocellulose obtained by subjecting lignocellulose to delignification, but may be lignocellulose not subjected to delignification.

  More specifically, lignocellulose is delignified with an organic acid such as peracetic acid or acetic acid in advance, and then the remaining holocellulose (cellulose + hemicellulose) is hydrolyzed by a pressurized hydrothermal method to obtain a monosaccharide (6 charcoal). Sugars and pentoses) and their hydrolysates containing oligosaccharides (cello-oligosaccharides, xylooligosaccharides, etc.) and uronic acid, etc. The hydrolyzate is converted to acetic acid by fermenting with acetic acid.

  Since peracetic acid is an organic acid, the degree of inducing polycondensation of lignin, that is, macromolecularization, like sulfuric acid is low, and lignin can be separated and recovered in a relatively low molecular state. This makes it possible to effectively use the lignin as a heat source and not as a polymer material having higher added value. In addition, peracetic acid and acetic acid used in the delignification process and remaining on the holocellulose side act as a catalyst in the next pressurized hot water process as it is, and not only greatly improve the hydrolysis efficiency of holocellulose, but also Together with acetic acid obtained in the fermentation process.

  Next, the present invention will be described in more detail with and without delignification treatment.

  After pulverizing the construction waste mainly consisting of rice straw, bagasse or conifers to a particle size of about 5mm, water is added to form a 1-5% lignocellulose slurry. When this lignocellulose is subjected to pressurized hydrothermal treatment without delignification treatment, part of the lignin is hydrolyzed to phenylpropane (C3-C6) structural units, and acetic acid is removed from the propyl side chain of the residue. Organic acids such as can be produced. If this is separated and recovered, the yield of organic acids such as acetic acid can be improved.

  In the process of delignifying lignocellulose and converting the remaining holocellulose to sugar, first, peracetic acid is added to the lignocellulose slurry to a concentration of 1 to 5% and then at 90 ° C. for 30 to 60 minutes. Cook lignocellulose. As a result, most of the coniferous lignin composed of guaiacyl-type lignin which is not easily reduced in molecular weight is removed (in the case of hardwood lignin, delignification is easier than coniferous, and 15 to Can be removed by cooking for about 20 minutes). On the other hand, in the case of using acetic acid, the delignification rate is 90% or more by cooking at 180 ° C for 3-4 hours in 90% concentration of acetic acid (Source: Wood Component Integrated Research Results, 1990). The lignin that has been reduced in molecular weight and dissolved by such a method is separated from the solid content (holocellulose) as a black liquor, and can be used as a polymer raw material after removing moisture. It is also possible to delignify lignocellulose, hydrolyze the resulting lignin to phenylpropane (C3-C6) structural units, and generate organic acids such as acetic acid directly from the propyl side chain of the residue It is. Here, holocellulose means a carbohydrate composed of cellulose and residual hemicellulose, and does not necessarily mean all hemicelluloses contained in lignocellulose in the original definition of holocellulose.

When the first-stage pressurized hot water of 140 to 230 ° C. is passed through the holocellulose separated from the lignin in this manner, the hemicellulose constituting sugar is passed, and then the second-stage pressurized hot water of 230 ° C. to 300 ° C. is passed. Cellulose constituent sugar can be separated and recovered. In the case of batch type treatment, peracetic acid used in the delignification step and remaining on the holocellulose side acts as a catalyst in this pressurized hot water treatment as it is, and hydrolysis at a lower temperature and treatment time becomes possible. In addition, the glucose yield can be improved (particularly effective during batch processing). Specific effects are as shown in Table 1.

  The purpose of delignification with organic acid in advance is to increase the added value of the polymer raw material by not polycondensing lignin as described above, and if the degradation product derived from lignin is later acetic acid fermentation by microorganisms Inhibiting the inhibitory factor is to remove the inhibitory factor in advance.

  The present invention involving a process of hydrolyzing holocellulose delignified with peracetic acid or acetic acid with pressurized hot water is particularly specialized in ethanol production from woody biomass and emphasizes practical aspects. In order to maximize the effects of indirect bioethanol process (partial sugars and part of lignin ⇒ acetic acid ⇒ ethanol) different from conventional yeast fermentation (C6 sugar ⇒ ethanol), instead of the enzymatic saccharification method that yields monosaccharides, It is assumed that an organic acid such as formic acid, lactic acid or acetic acid, or an oligosaccharide or uronic acid containing a monosaccharide is obtained by hydrolysis with pressurized hot water and subsequent fragmentation reaction. Another major feature is the use of peracetic acid and acetic acid as a catalyst with a view to reducing the hydrolysis temperature and further the fragmentation reaction temperature, and effective utilization thereafter.

  When batch-type pressurized hydrothermal treatment is performed on holocellulose, the quinone compounds described in JP-A-2005-40025, particularly p-benzoquinone, are added to the reaction system to reduce oligosaccharide reducing ends and glucose. By effectively suppressing side reactions (secondary decomposition) such as fragmentation, hydrolysis of holocellulose can be further promoted, and the yield of sugars such as glucose can be significantly increased. The addition amount of the quinone compound is most preferably in the range of 0.05 to 1 wt% with respect to the total reaction system.

Basically, a culture method of Clostridium thermoaceticum, a homo-acetic acid-fermenting bacterium starting from hexose or pentose monosaccharide described in Patent Document 1 or Clostridium formicoaceticum, starting from lactic acid. And its metabolic pathway (see the following reaction formulas _{(VII), (VIII), (IX)} ). Clostridium bacteria have the ability to assimilate a wide range of sugars, and can convert 6- and 5-carbon oligosaccharides (such as cellooligosaccharides and xylooligosaccharides) or uronic acid into acetic acid. Therefore, much more sugar substrates can be converted to acetic acid without using gene recombination technology as compared with conventional ethanol fermentation using yeast or the like. It is sufficient to hydrolyze holocellulose to oligosaccharide instead of monosaccharide in the pressurized hydrothermal treatment in the previous step, or even if it is mixed with monosaccharide, there is no problem, and holocellulose is used with expensive enzyme (cellulase) Thus, it is not necessary to saccharify all the way to monosaccharides.

Esterification and hydrocracking step This is in accordance with a known method described in JP 2002-537848 A (see the following reaction formulas (X), (XI), (XII), (XIII)). In addition, the esterification process of (X) and (XII) can also be performed without a catalyst by using supercritical water as Example 3 mentioned later.

  The lignocellulose can be recovered in a low molecular weight state without polycondensation by subjecting it to delignification treatment with an organic acid such as peracetic acid before being subjected to pressurized hot water treatment. In addition to achieving high added value, when the lignin-derived degradation product inhibits acetic acid fermentation by microorganisms later, the inhibitory factor can be removed in advance.

  By delignining lignocellulose, it is possible to recover only holocellulose (cellulose + hemicellulose) from lignocellulose. If this is treated with pressurized hot water, the lignin-derived pyrolysis product is not mixed. Is obtained.

  By subjecting lignocellulose to delignification, since there is no lignin embedding holocellulose, hydrolysis efficiency with pressurized hot water is improved.

  The organic acid such as peracetic acid remaining on the holocellulose side works as a catalyst in the pressurized hot water treatment, and can improve the hydrolysis efficiency of the holocellulose.

  When hydrolyzing holocellulose by a pressurized hydrothermal method, by adding a quinone compound, hydrolysis of holocellulose can be promoted, and thermal decomposition of saccharides can be suppressed to improve sugar yield.

  Next, in order to describe the present invention specifically, examples of the present invention will be given. However, the present invention is not limited to these.

[Example 1]
Hydrolysis of lignocellulose with pressurized hot water Treated hardwood beech wood flour (through 18 mesh) with ethanol: benzene (1: 2) mixture, dried in 105 ° C oven for 24 hours and used did. In order to study each treatment condition for selectively and efficiently hydrolyzing hemicellulose and cellulose, using a semi-circulating two-stage pressurized hot water treatment apparatus shown in FIG. 1, beech wood flour (1.0 g) One-stage pressurized hot water treatment (10 ml / min) was performed for 15 minutes at various treatment temperatures (170, 190, 210, 230, 250, 270, 290 ° C.). The pressure was set to 10 MPa (constant). In this device, pressurized hot water heated separately by two preheater heaters (Heater-1 and 2) can be fed sequentially into the reaction tube by switching the valve, which makes beech wood flour (1.0 g) The two-stage pressurized hot water treatment (10 ml / min) can be efficiently performed under different temperature conditions, and the reaction is stopped using a cooling tube. Because of external cooling, the solubilized product is not diluted, and the product (saccharide) can be recovered at a relatively high concentration.

  FIG. 2 shows the production amounts of (A) xylooligosaccharide and xylose, (B) cellooligosaccharide and glucose, (C) furfural and (D) 5-hydroxymethylfurfural (5-HMF) at each treatment temperature. Xylooligosaccharides and xylose derived from xylan, which are the main hemicellulose components of beech wood flour, obtained the highest yield at 230 ° C, and cellulooligosaccharides and glucose derived from cellulose at 270 ° C. In addition, furfural, which is considered to be a hemicellulose-derived pentose superdegradation product, rapidly increases in production when it exceeds 230 ° C, and the amount of 5-HMF, which is considered to be a cellulose-derived hexose sugar overdegradation product, increases. Increased above 250 ° C.

Based on the above results, xylooligosaccharides and cellooligosaccharides were obtained in the highest yield, and the processing conditions that minimize the excessively decomposed furfural and 5-HMF were [230 ml at a flow rate of 10 ml / min. ℃ / 10 MPa (15 minutes), 270 ℃ / 10 MPa (15 minutes)]. Under this condition, the beech wood powder slurry was subjected to two-stage pressurized hot water treatment, and the resulting decomposition product was divided into components obtained by the first-stage treatment and the second-stage treatment. Further, Table 2 shows the results of expressing hemicellulose-derived and cellulose-derived saccharides and saccharide hyperdegradation products in terms of weight percent based on wood flour.

  The constituent sugars of glucuronoxylan, the main hemicellulose component of beech wood flour, are xylose (2) and glucuronic acid (3), and xylooligosaccharide (1), xylose (2) and glucuronic acid (3) obtained by this treatment ) Total yield was about 15% on a wood flour basis and about 59% on a glucuronoxylan basis. 99% of them were obtained from the first stage treatment. On the other hand, cellooligosaccharide (8) and glucose (9), which are thought to be derived from cellulose, fructose (10), which is an isomer of glucose, and levoglucosan (11), which is a dehydrated product, are 34% based on wood flour and cellulose based. 71% were obtained, 86% of which was obtained from the second stage treatment.

  Further, FIG. 3 shows that xylooligosaccharides derived from glucuronoxylan (1), xylose (2), glucuronic acid (3) and acetic acid (18), which are the main hemicellulose components of beech wood flour, and cellooligosaccharides derived from cellulose (8 ), Glucose (9), fructose (10), which is an isomer of glucose, and levoglucosan (11), which is a dehydrated product, and the relationship between the treatment conditions. This result clearly shows that 230 ° C. (first stage treatment) is a selective hydrolysis region of hemicellulose and 270 ° C. (second stage treatment) is a selective hydrolysis region of cellulose.

  In this example, 27% of the lignin-derived substance present on a wood flour basis was not analyzed, but 31% of the unidentified substance was present, and 14% of the sugar was decomposed. As a result of quantifying the residue after hydrolysis by the Klarson lignin method, almost 100% is lignin, which is about 2.8% based on wood flour and about 10% based on lignin. That is, it is considered that 100% of the polysaccharides and hemicelluloses as polysaccharides and about 90% of the lignin as an aromatic compound are solubilized by the pressurized hot water of this example. In this patent, it is not necessary to hydrolyze lignocellulose to monosaccharides, and oligosaccharides are expected to be substrates for acetic acid fermentation (Example 2) in the next step.

[Example 2-1]
Acetic acid fermentation for a wide range of sugars 1
Prepare the solutions 1, 2, and 3 shown below, sterilize and deaerate them at 120 ° C for 40 minutes in an autoclave, and then fill and mix 100, 25, and 15 ml of solutions 1 to 3, respectively. (50 ml in total) was inoculated with Clostridium thermoaceticum (ATCC39073 strain) and cultured in a CO ₂ atmosphere at 60 ° C. for 3 days to obtain a preculture solution.

Solution 4 is then prepared and sterilized and degassed in the same manner, and then filled into a 100 ml syringe vial with 5 ml of solution 4, 25 ml of solution 2, 15 ml of solution 3, and 5 ml of the above-mentioned pre-culture solution for a total of 50 ml. And acetic acid fermentation in a CO ₂ atmosphere at 60 ° C.

Solution 1: 10 g glucose, 100 ml distilled water
Solution 2: Yeast extract 5 g, cysteine hydrochloride monohydrate 0.25 g, ammonium sulfate 1 g, magnesium sulfate heptahydrate 0.25 g, ferrous ammonium sulfate hexahydrate 0.04 g, nickel chloride hexahydrate 0.24 mg, Zinc sulfate heptahydrate 0.29 mg, Sodium selenite 0.017 mg, Resazurin (1% solution) 0.1 ml, Distilled water 300 ml
Solution 3: sodium hydroxide 0.415 g, sodium hydrogen carbonate 5 g, dipotassium phosphate 4.4 g, potassium dihydrogen phosphate 7.5 g, distilled water 150 ml
Solution 4: 10 g of various sugars, 100 ml of distilled water (various sugars are glucose, xylose, mannose, galactose, arabinose, fructose and glucuronic acid)
A small amount of the culture solution is collected every 12 hours from each test section, and the results of analyzing the transition of various saccharides and their acetic acid concentrations by HPLC are shown in Fig. 4-1 (measured until 60 hours later). All sugars decreased with the passage of fermentation time, and acetic acid was produced accordingly. The acetic acid fermentation of mannose, galactose, arabinose, and glucuronic acid has a longer fermentation time than that of glucose, xylose, and fructose. However, as the fermentation progresses, sugars decrease and acetic acid is generated. The batch type acetic acid fermentation test revealed that various monosaccharides expected to be obtained from lignocellulose can be substrates for acetic acid fermentation by C. thermoaceticum, although there are differences in acetic acid fermentation time. In particular, glucose, xylose and fructose all produced about 8 g / l of acetic acid from 10 g / l of sugar, and the conversion efficiency was good at about 80%. Moreover, it has also been confirmed that hydrogen gas is generated in this acetic acid fermentation system, and the generated hydrogen can be used for the reaction in the next step (hydrocracking).

[Example 2-2]
Acetic acid fermentation for a wide range of sugars 2
Prepare the solutions 1, 2, and 3 shown below, sterilize and deaerate them at 120 ° C for 40 minutes in an autoclave, and then fill and mix 100, 25, and 15 ml of solutions 1 to 3, respectively. (50 ml in total) was inoculated with Clostridium thermocellum (ATCC27405 strain) and cultured in a CO ₂ atmosphere at 60 ° C. for 3 days to obtain a preculture solution.

Solution 4 is then prepared and sterilized and degassed in the same manner, and then filled into a 100 ml syringe vial with 5 ml of solution 4, 25 ml of solution 2, 15 ml of solution 3, and 5 ml of the above-mentioned pre-culture solution for a total of 50 ml. And acetic acid fermentation in a CO ₂ atmosphere at 60 ° C.

Solution 1: 5 g of cellobiose, 200 ml of distilled water
Solution 2: Yeast extract 4.5 g, glutathione 0.25 g, ammonium sulfate 1.3 g, magnesium sulfate hexahydrate 0.13 g, ferrous sulfate heptahydrate 0.0011 g, calcium chloride dihydrate 0.13 g, resazurin 0.001 g, distilled 500 ml of water
Solution 3: potassium dihydrogen phosphate 1.43 g, dipotassium phosphate trihydrate 7.2 g, sodium glycerophosphate 6 g, distilled water 300 ml
Solution 4: Cellulose, cellohexaose or cellobiose 5 g, distilled water 50 ml
A small amount of the culture solution is collected every 12 hours from each test section, and the results of analyzing the transition of acetic acid concentration by HPLC are shown in Fig. 4-2 (measured until 120 hours later). Acetic acid was produced over the course of fermentation time when any of the polysaccharides cellulose and oligosaccharides cellohexaose and cellobiose were used. Although there are differences in acetic acid fermentation time, the typical polysaccharides expected to be obtained from lignocellulose, cellulose and cellooligosaccharides, which are oligosaccharides, can all be substrates for acetic acid fermentation by C. thermocellum. It was found by fermentation test. This indicates that it is not necessary to hydrolyze lignocellulose to a monosaccharide in the pressurized hot water treatment described in Example 1.

[Example 2-3]
Acetic acid fermentation of formic acid and lactic acid In the method described in Example 2, 10 g of various saccharides in solution 4 were subjected to acetic acid fermentation with formic acid and lactic acid using 1 g of formic acid or lactic acid.

  A small amount of culture solution was collected from each test section, and the results of HPLC analysis of the transition of formic acid, lactic acid and acetic acid concentrations are shown in FIG. 4-3 (measured until 120 hours later). (a) shows the results with formic acid, but it can be seen that lactic acid and acetic acid are produced by fermentation. However, acetic acid increased with the passage of fermentation time, but lactic acid tended to decrease as fermentation progressed. (b) shows the result with lactic acid, but it can be seen that lactic acid is converted to acetic acid. From these results, it is considered that formic acid and lactic acid by-produced by acetic acid fermentation by C. thermocellum described in Example 2-2 can all be converted to acetic acid when C. thermoaceticum is used in combination.

[Example 2-4]
Acetic acid fermentation in a mixed system of C. thermoaceticum and C. thermocellum By the method described in Example 2, a preculture solution of C. thermoaceticum (hereinafter referred to as culture solution 1) was prepared. A pre-culture solution of C. thermocellum (hereinafter referred to as culture solution 2) was prepared by the method described in Example 2-2.

Solution 5 is then prepared, sterilized and degassed into a syringe vial of 100 ml capacity, 5 ml of solution 5, 25 ml of solution 2 described in Example 2, 15 ml of solution 3, and 2.5 ml of the aforementioned preculture 1 A total of 50 ml was filled with 2.5 ml of the preculture 2 and subjected to acetic acid fermentation in an N ₂ atmosphere at 60 ° C.

Solution 5: 10 g cellulose, 100 ml distilled water
A small amount of the culture solution is collected from each test section, and the results of analyzing the transition of acetic acid concentration by HPLC are shown in Fig. 4-4 (measured until 120 hours later). Acetic acid was produced over the course of the fermentation time. In addition, lactic acid as a by-product was completely consumed over the course of fermentation time. The acetic acid conversion pathway of this mixed fermentation system can be understood as shown in FIG. C. thermocellum, which can use cellulose as a substrate, converts cellulose into glucose and further converts into acetic acid, formic acid, lactic acid, ethanol and a small amount of hydrogen and carbon dioxide (Example 2-2), but coexisting C In order to convert glucose, formic acid and lactic acid produced by .thermoaceticum into acetic acid (Examples 2 and 2-3), it is considered that cellulose could be converted into acetic acid with high efficiency. C. thermoaceticum and C. thermocellum mixed fermentation systems can be said to be excellent fermentation systems capable of converting a wide range of sugars into acetic acid.

[Example 3]
Non-catalyzed esterification of acetic acid After injecting ethanol and acetic acid at a molar ratio of 1: 1 to an Inconel 5 ml batch type reaction tube, the reaction tube was sealed, and then treated in a tin bath at 270 ° C. for 20 minutes. The obtained processed product was analyzed by HPLC, and it was confirmed that ethyl acetate was produced. The reaction system was a non-catalytic system to which no catalyst was added, thereby confirming the esterification reaction with supercritical ethanol. The results of the HPLC analysis are shown in the HPLC chromatogram in FIG. 5 (HPLC chromatogram processing conditions: 270 ° C./20 min / molar ratio 1: 1, column: Ultron PS-80P, mobile phase: water 1 ml / min, detector) : RID).

[Example 4]
Conversion of ethyl acetate to ethanol by catalytic hydrogenation Copper-zinc catalyst (Cu / Zn = 48/44, w / w, 30-100 mesh, 1.7 g) was packed into an Inconel reaction tube and N ₂ (60 ml / min) /250°C/0.1 MPa / 2 hours after calcination, change the atmosphere to H ₂ (30 ml / min) + N ₂ (30 ml / min), H ₂ (60 ml / min) Heat treatment was performed for a period of time to activate the catalyst. Thereto, ethyl acetate (0.01 to 0.11 ml / min, ethyl acetate / H ₂ = 1: 4) was introduced with a liquid feed pump to perform catalytic hydrogenation of ethyl acetate. Quantitative analysis of ethanol and ethyl acetate was performed on the aggregated and recovered liquid product using GC [GC conditions: column: Shimadzu CBP-5 (25 m x 0.25 mmΦ), carrier gas: He (1.5 ml / min ), Column temperature: 40 ° C. (0-12 min), 40 → 150 ° C. (12-15 min), injector temperature: 250 ° C.]. As shown in FIG. 6, the selectivity of the reaction was very high, and almost no components other than ethyl acetate and ethanol were detected in the recovered liquid. The relationship between the reaction temperature and pressure and the ethanol yield is shown in FIG. 7. As the reaction pressure increased, the conversion efficiency to ethanol was improved, and it was found that a reaction temperature of 250 ° C. was appropriate. The conversion efficiency to ethanol under the condition of 250 ° C./2 MPa was 80% or more, and the conversion efficiency when using a catalyst of 30 mesh or less under the same conditions was 99.7%.

[Example 5]
Ethanol production by one-pot catalytic hydrogenation from acetic acid In Example 3, it was shown that ethyl acetate was produced without catalyst by keeping acetic acid and ethanol at high temperature and high pressure. Since the conditions for catalytic hydrogenation of ethyl acetate in Example 4 are high temperature and high pressure conditions, it is possible to continuously perform esterification of acetic acid and catalytic hydrogenation in one pot. From such a background, the apparatus shown in FIG. 8 was devised. That is, first, esterification proceeds by maintaining acetic acid and ethanol at a high temperature and high pressure, and then the catalyst layer [copper-zinc catalyst (Cu / Zn = 48/44, w / w, 1.7 g) on the downstream side. ). As a result of experiments in such a system, it was confirmed that the conversion to ethanol proceeded.

Schematic diagram of a semi-circulating two-stage pressurized hot water treatment device (A) xylose and xylooligosaccharides, (B) glucose and cellooligosaccharides, (C) furfural, (D) 5-HMF at each temperature in one-stage pressurized hydrothermal treatment using a semi-flow type device Change in yield (% by weight based on wood flour) Two-stage pressurized hot water treatment (pressure: 10 MPa) temperature conditions and decomposition products (sugars derived from glucuronoxylan and cellulose) yield (% by weight based on wood flour) Changes in sugar and acetic acid concentrations during acetic acid fermentation using Clostridium thermoaceticum for each sugar Concentration change of various organic acids in acetic acid fermentation using polysaccharide and oligosaccharide Clostridium thermocellum Changes in concentrations of various organic acids in acetic acid fermentation of formic acid and lactic acid using C. thermoaceticum Changes in concentration of various organic acids during acetic acid fermentation of cellulose by C. thermoaceticum and C. thermocellum mixed systems Acetic acid conversion pathway in C. thermoaceticum and C. thermocellum mixed fermentation systems HPLC chromatogram showing esterification reaction Example of GC analysis results after catalytic hydrogenation treatment (250 ℃ / 2MPa) Effects of reaction temperature and pressure on catalytic hydrogenation of ethyl acetate Overview of one-pot catalytic hydrogenation equipment from acetic acid

Claims (6)

Hydrolyzing lignocellulose by pressurized hot water treatment ;
A step of converting the degradation products of lignocellulose produced by the hydrolyzing step in acetic acid,
Obtaining a ethyl acetate by esterifying the acetic acid,
Obtaining a ethanol the ethyl acetate by hydrogenolysis,
Have
The step of converting the degradation product of lignocellulose into acetic acid coexists with Clostridium thermocellum and Clostridium thermoaceticum to decompose the oligosaccharide contained in the degradation product of lignocellulose by the Clostridium thermocellum, and the Clostridium thermoaceticum method for producing an alcohol you convert other than the oligosaccharide acetate of decomposition products and decomposition products of the lignocellulose of the oligosaccharide by. The method for producing an alcohol according to claim 1 , wherein the degradation product of lignocellulose includes hemicellulose-derived saccharides, cellulose-derived saccharides, hyperdegradation products thereof, organic acids, and degradation products derived from lignin. . The hydrolyzing step includes a pressurized hot water treatment, a pre-treatment for decomposing hemicellulose with 140-230 ° C. pressurized hot water, and a pressurized heat in the range of 230-300 ° C. and higher than the pre-treatment. The method for producing an alcohol according to claim 1 or 2, which is carried out in two stages of a subsequent process of decomposing cellulose with water . The method for producing an alcohol according to any one of claims 1 to 3 , wherein a delignification treatment for removing lignin from the lignocellulose is performed before the pressurized hot water treatment . The method for producing an alcohol according to claim 4, wherein the delignification treatment is performed using peracetic acid or acetic acid . The method for producing an alcohol according to claim 4, wherein a quinone compound is added in the hydrolysis step .

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